PoDPBT, a BAHD acyltransferase, catalyses the benzoylation in paeoniflorin biosynthesis in Paeonia ostii

PoDPBT, an O‐benzoyltransferase belonging to the BAHD family, can catalyze the benzoylation of 8‐debenzoylpaeoniflorin to paeoniflorin. PoDPBT is the first enzyme demonstrated to be involved in the modification stage of paeoniflorin biosynthesis. DFGGG, a new DFGWG‐like motif, was revealed in the BAHD family. The transcriptome database provides a resource for further investigation of other enzyme genes involved in paeoniflorin biosynthesis.

Paeoniflorin, a monoterpene bicyclic glycoside, is a very important medicinal compound with multiple pharmacological activities such as antidepression, antithrombosis, immunoregulation, and neuroprotection (Zhang et al., 2022;Zhao et al., 2020). Moreover, paeoniflorin is only found in Paeoniaceae plants and is considered as the characteristic compound of Paeoniaceae (Zhang et al., 2019). However, its biosynthesis pathway remains to be fully elucidated. Benzoyl-CoA: benzyl alcohol Obenzoyltransferases (BEBTs) belong to the BAHD acyltransferase family, and these enzymes possess a high affinity for benzoyl-CoA as the acyl donor and can utilize different aromatic alcohols as the acyl acceptors (Chedgy et al., 2015). 8-Debenzoylpaeoniflorin, one of derivatives of paeoniflorin, is also widely distributed in Paeoniaceae. Unlike paeoniflorin, 8-debenzoylpaeoniflorin has a hydroxyl rather than a benzoyl group at the C8 position, and thus its structure is similar to the aromatic alcohol. Therefore, we speculated that an O-benzoyltransferase existing in Paeoniaceae plants could utilize benzoyl-CoA and 8-debenzoylpaeoniflorin as substrates to synthesize paeoniflorin (Zhang et al., 2022). This enzyme would be designated as a benzoyl-CoA: 8debenzoylpaeoniflorin 8-O-benzoyltransferase (DPBT).
We previously found the paeoniflorin contents in the leaf of Paeonia ostii were significantly correlated with the plant developmental stages (Zhang et al., 2019). To screen the genes encoding O-benzoyltransferase involved in paeoniflorin biosynthesis, RNA samples from leaves were individually harvested from the budding stage (S3), bud expanding stage (S4) and flowering stage (S5) of P. ostii ( Figure S1), and then sequenced on a BGISEQ-500 platform (Figure 1a). The reliability of RNA-Seq data was verified by qRT-PCR ( Figure S2 and Table S1). Twenty-three O-benzoyltransferase gene candidates were identified based on the gene annotations (Table S2) with different expression patterns at different development stages (Figure 1b). Among them, the expression levels of Pos.gene26002, Pos.gene30573, Pos.gene35667, Pos.gene79215, and Pos.gene81370 were highly correlated (coefficient >0.9) with the paeoniflorin content. However, except for Pos.gene30573, the other four genes had the FPKM values below 1.00 across all the three development stages (Table S2). Therefore, we focused on the role of Pos.gene30573 in converting 8-debenzoylpaeoniflorin to paeoniflorin and named it as PoDPBT.
We then examined the expression pattern of PoDPBT in different leaf development stages of P. ostii by qRT-PCR. PoDPBT was highest expressed in S3, followed by lower levels in S4 and S5, which was consistent with the accumulation of paeoniflorin ( Figure 1c). Our previous study indicated paeoniflorin levels varied in different P. ostii organs (Zhang et al., 2019). The expression patterns were also checked, and the results showed the expression of PoDPBT was highest in the stem containing the most paeoniflorin ( Figure 1d). These results further confirmed the correlation between the expression levels of PoDPBT and the paeoniflorin content, suggesting that PoDPBT is involved in the paeoniflorin biosynthesis.
Sequence analysis showed PoDPBT encodes a predicted 453amino acid protein of 49.91 kD. PoDPBT contains the HXXXD motif that is highly conserved for BAHD members, and another conserved region DFGWG motif is slightly altered with glycine instead of tryptophan. In addition to these two conserved motifs, YPLAGR and QVTRLKCGG motifs were also identified ( Figure 1e) (Tuominen et al., 2011). These results indicated PoDPBT is a member of BAHD acyltransferase family. The phylogenetic tree showed PoDPBT is arranged in BAHD clade V-i (Figure 1f), and members of this clade catalyse the formation of both volatile and non-volatile esters (Chedgy et al., 2015). PoDPBT has a close relationship with CbBEBT, NtBEBT (D'Auria et al., 2002), VhBEBT (Togami et al., 2006), and PtBEBT (Chedgy et al., 2015). These results suggested PoDPBT could utilize benzoyl-CoA and 8-debenzoylpaeoniflorin to synthesize paeoniflorin (Figure 1g).
PoDPBT was expressed in Escherichia coli, and the purified protein size was consistent with the expected 49.91 kD (Figure 1h). In vitro enzyme assays were performed with benzoyl-CoA and 8-debenzoylpaeoniflorin. The UPLC-MS results showed the new peak from experimental samples had the same retention time and MS spectrum as the authentic paeoniflorin standard (Figure 1i This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. To further validate the function of PoDPBT in vivo, we used a virus-induced gene silencing system to suppress the expression of PoDPBT in P. ostii leaves. A tobacco rattle virus 2 (TRV2) vector was constructed with green fluorescent protein (GFP) as a reporter. Green fluorescent spots were observed under blue light in the leaves infiltrated with TRV2-GFP and TRV2-PoDPBT-GFP (Figure 1l) but not in the normal plants. The expression level of PoDPBT in the leaves infiltrated with TRV2-PoDPBT-GFP was significantly lower than those of the normal plants and TRV2-GFP lines, and there was no significant difference between the latter two lines (Figure 1m). The lowest content of paeoniflorin was also detected in the leaves of TRV2-PoDPBT-GFP lines (Figure 1n), and the remaining paeoniflorin is produced due to leftover PoDPBT. These results further confirmed the role of PoDPBT in the paeoniflorin biosynthesis.
Overall, PoDPBT, an O-benzoyltransferase belonging to the BAHD family, was identified and functionally characterized, and this enzyme was able to catalyse the benzoylation of 8debenzoylpaeoniflorin to paeoniflorin as a single product. PoDPBT is the first enzyme involved in the modification stage of paeoniflorin biosynthesis. Other enzymes such as hydroxylase and glycosyltransferase involved in this stage have been predicted (Zhang et al., 2022), and these enzymes will be characterized successively based on the research strategy established in this study. These results will lay the foundation for the complete elucidation of paeoniflorin biosynthesis pathway.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.

Figure S1
Leaves of Paeonia ostii at the budding stage (S3), bud expanding stage (S4) and flowering stage (S5). Figure S2 Analysis and validation of DEGs between the budding stage and flowering stage leaves. Table S1 The list of 19 DEGs validated by qRT-PCR. Table S2 The list of 23 benzoyltransferase gene candidates. Appendix S1 Methods used in this study. UPLC profile of the product generated by empty vector enzyme with 8-debenzoylpaeoniflorin (i) and the product of PoDPBT incubated with 8debenzoylpaeoniflorin (j); Mass spectrum data of the product of PoDPBT enzyme assay (k). (l-n) Silencing effect of PoDPBT on paeoniflorin production. Image of P. ostii leaves infected with TRV2-GFP and TRV2-PoDPBT-GFP at 6 days after infiltration under blue light, and with normal plants as control (CK) (l); Expression levels of PoDPBT in different treatment leaves by qRT-PCR (m); Paeoniflorin contents in different treatment leaves (n). Asterisks indicate statistically significant differences (**P < 0.01, Student's t-test).